NaCl exposure results in increased expression and processing of IL-1β in Meniere’s disease patients

Meniere’s disease (MD) is a chronic disease that causes episodic vertigo, fluctuating hearing loss, and aural fullness, initially managed by dietary salt reduction, and use of diuretics. Our prior research in autoimmune inner ear disease (AIED) demonstrated that in peripheral blood mononuclear cell (PBMC) from corticosteroid-resistant AIED patients, increased production, processing and release of interleukin-1β (IL-1β) is observed and hearing could be improved with use of anakinra, an interleukin-1 receptor antagonist. We have further identified that in these AIED patients, IL-1β is uniquely processed to a 28 kDa pro-inflammatory product by caspase-7. In the present study, we characterize the production, processing and release of the pro-inflammatory cytokines IL-1β and IL-6 from PBMC of MD (n = 14) patients in response to sodium chloride (NaCl), and determined the effect of the diuretic triamterene-hydrocholothiazide (T-HCTZ), or anakinra in these patients. We observed that PBMC cultured with NaCl from MD patients show processing of IL-1β to the 28 kDa product, and that this product is abrogated with T-HCTZ. Our observations are consistent with other autoimmune diseases where high concentrations of NaCl caused release of pro-inflammatory cytokines and may provide further insight as to the mechanism of disease progression in MD patients.


Results
We determined effects of NaCl in the ability to induce pro-inflammatory cytokine production in PBMC from a small cohort of definite MD patients and control subjects. Patient demographics of those included in these studies are shown in Table 1. Notably, controls recruited for these studies reported no personal or family history of MD, vertigo, hearing loss or autoimmune disease. All MD patients had active disease at the time of recruitment. A subset of MD patients was given corticosteroids at the time of recruitment for a decline in hearing ( Table 1). The majority of MD patients did not respond (Table 1). Concomitant vestibular migraine was observed in 3 of the 14 patients with MD (Table 1). www.nature.com/scientificreports/ NaCl treatment results in dose-dependent increase in IL-1β and IL-6 m RNA levels in the PBMCs of MD patients. We determined the dose dependent effect of NaCl stimulation in PBMC by Quantitative Real-Time PCR (Q-RT-PCR) from MD patients (n = 10) and control subjects (n = 10). A dose-dependent increase in IL-1β and IL-6 mRNA levels were observed in MD patients with maximal expression observed at 80 mM NaCl (Fig. 1a,b). In controls, NaCl stimulation of PBMC resulted in minimal mRNA expression of IL-1β and IL-6 ( Fig. 1a,b). Statistical analysis by one-way ANOVA resulted in p = 0.0005 and p = 0.0035. For IL-1β and IL-6 respectively. Applying a Bonferroni's multiple comparison test still indicated a significant effect (p < 0.05) for comparison between 80 mM NaCl treatment in controls vs. 80 mM NaCl treatment in MD patients for both IL-1β and IL-6 ( Fig. 1a,b).

NaCl treatment results in a dose-dependent increase in IL-1β and IL-6 release in the PBMCs of MD patients.
To determine whether the transcriptional induction of IL-1β and IL-6 resulted in increased circulating pro-inflammatory cytokines in the presence of sodium chloride, we performed a sandwich ELISA on MD patients (n = 12) and controls (n = 12). NaCl stimulation of PBMC again demonstrated a dose-dependent increase in IL-1β and IL-6 release in MD patients, (Fig. 2a,b). Statistical analysis by one-way ANOVA resulted in p = 0.0001 and p = 0.0001 for IL-1β and IL-6 respectively. Applying a Bonferroni's multiple comparison test still indicated a significant effect (p < 0.05) for comparison between 80 mM NaCl treatment in controls vs. 80 mM NaCl treatment in MD patients for both IL-1β and IL-6 ( Fig. 2a,b).
NaCl treatment results in a dose-dependent increase in generation of 28 kDa IL-1β fragment in MD patients and control subjects fail to generate 28 kDa band of IL-1β. When PBMCs isolated from MD patients were stimulated with NaCl and protein isolated for Western blot analysis, the 28 kDa fragment of IL-1β was observed in a dose-dependent manner in response to increasing NaCl in MD patients (n = 5) when compared to normal healthy subjects (n = 5). A representative blot shown in Fig. 3a. Notably, Potassium chloride (KCl) had no effect on IL-1 β induction, arguing that the observed induction of IL-1 β in response to NaCl is specific and cannot be ascribed to all salts (Fig. 3b). www.nature.com/scientificreports/ Triamterene-hydrochlorothiazide (T-HCTZ) inhibited the NaCl-induced 28 kDa band of IL-1β in MD patients. Triamterene-hydrochlorothiazide (T-HCTZ) inhibited the observed NaCl induction of the 28 kDa band of IL-1β in a dose dependent manner in MD patients. PBMCs were treated with increasing concentrations of T-HCTZ (10 -8 to 10 -6 M) in combination with 80 mM NaCl. T-HCTZ at the concentration of 10 -6 M was found to be most effective in mitigating the NaCl effect ( Fig. 4a,b), the blots are representative of two of the nine blots performed). Statistical analysis by one way ANOVA resulted in p = 0.0011. Applying a Bonferroni's multiple comparison test still indicated a significant effect (p < 0.05) for comparison of T-HCTZ (10 −6 M) and NaCl with NaCl alone (Fig. 4c).  www.nature.com/scientificreports/

Discussion
We identified that high dietary NaCl diet triggers inflammation through the production and release of the proinflammatory cytokines IL-1β and IL-6 from PBMC, resulting in the clinical exacerbation of MD. The induction of pro-inflammatory cytokine transcription, translation and release was optimal at 80 mM of NaCl and specific, as KCl did not similarly induce IL-1β. The 40-80 mM concentration of NaCl has been used across many studies 40,41 , and is believed to represent the physiological concentration range of NaCl in the interstitium and lymphoid tissue 21,42 . In a study done by Shapiro and Dinarello 41 , a range of 40 to 80 mM NaCl was optimal for induction of another proinflammatory cytokine, IL-8, in PBMCs of healthy donors. We further demonstrated that the PBMCs of MD patients respond differently to dietary NaCl than the PBMCs of normal healthy controls: specifically, MD patients process IL-1β to the pro-inflammatory 28 kDa form in a dose-dependent manner in response to NaCl. Vestibular migraine (VM) may co-exist with MD or it may mimic MD, as clinical symptoms overlap. Recent studies have demonstrated that these two entities can be distinguished by their cytokine profile, where vestibular migraine patients express significantly less IL-1β than MD patients 43,44 . In our studies, out of 14 MD patients studied, only 3 patients had both VM and MD, whereas 11 had MD alone. The plasma and conditioned supernatant values of these two groups were similar: VM + MD was 1.2 pg/ml and 2.56 pg/ml whereas MD was 1 pg/ml and 3.51 pg/ml, although we did not examine patients with VM alone which explains why a substantial difference was not observed. All patients were recruited during a period of active disease flare. In 9 of 14 patients, steroids were given immediately following recruitment because of an observed hearing decline and consistent with previous reports, the majority did not respond to corticosteroids. We also examined whether there was a difference in NaCl induced IL-1β release between these patients with a hearing decline necessitating corticosteroid use and those that did not (Table 1). Our results indicated that a nominal 1.4-fold difference in IL-1β release in response to NaCl in patients that necessitated corticosteroid therapy.
Resident macrophages have been identified in the stria vascularis and endolymphatic sac 40,45 which are functionally similar to monocytes and microglia. Blockade of sodium channels (especially Nav1.6) in microglia attenuated the release of LPS-stimulated IL-1β 46 . Macrophages monitor tissue osmolarity and induce inflammation through NLRP3 47 . A drop in intracellular potassium and to a lesser degree an increase in intracellular sodium could enhance the activity of NLRP3 47 . A high salt diet has been associated with ENaC-dependent NLRP3 activation in dendritic cells in a murine model of hypertension 48 . It has been shown in the respiratory tract and kidneys that pro-inflammatory cytokines down-regulates expression of ENaC and Na/K ATPase, and interferes with sodium transport 49 . The diuretic triamterene is a known inhibitor of the Na/K-ATPase in the kidney plasma membrane 10,50 and of ENaC 51 therefore may exert an effect on multiple Na/K-ATPases, including those in immune cells. To address this question, we isolated monocytes from a patient with autoimmune disease and stimulated with NaCl in the presence of either T-HCTZ or ouabain ((an inhibitor of the Na/K-ATPase) www.nature.com/scientificreports/ (Supplemental Fig. 1)). T-HCTZ inhibited generation of the 28 kDa IL-1 product suggesting ENaC is involved in the processing of IL-1, whereas ouabain did not. However, the combination of ouabain and T-HCTZ abrogated expression of both the 28kD and the pro-IL-1 bands (33/31 kDa), suggesting a synergistic effect on IL-1 expression. Future studies determining the relative contributing roles of ENaC and the Na/K-ATPase in both MD and AIED may provide interesting new therapeutic targets for intervention in these diseases. To our knowledge, this is the first study investigating the effects and correlating the role of high dietary NaCl diet in triggering inflammation through the production and release of pro-inflammatory cytokines such as IL-1β and IL-6 and induction of 28 kDa IL-1 band in MD patients. Our results indicated that diuretic T-HCTZ was effective in inhibiting 28 kDa IL-1β band in PBMC of MD patients at concentration of 10 -6 M. The observation that T-HCTZ inhibited NaCl-induced IL-1β expression may provide additional mechanistic information in combination with NaCl at concentration 80 mM, along with LPS as positive control and kept untreated. The samples were subjected to Western blotting using IL-1β antibody. β-actin was used as a control for total protein. The representative blots for two out of nine MD patients are shown in the figure. (b) NaCl failed to induce IL-1β in control subjects. PBMCs from healthy controls were treated with increasing concentration of T-HCTZ in combination with NaCl at concentration 80 mM, along with LPS as positive control and kept untreated. The samples were subjected to Western blotting using IL-1β antibody. β-actin was used as a control for total protein. The representative blots for two out of ten control subjects are shown in the figure. (c) NaCl failed to induce IL-1β in control subjects. The histogram shows the quantitative densitometry of 28 kDa protein of IL-1β (fold over control) normalized over actin expression from ten control subjects and nine different MD patients. The data is shown as mean ± SEM.). Statistical analysis by one-way ANOVA resulted in p = 0.0011. Applying a Bonferroni's multiple comparison test still indicated a significant effect (p < 0.05) for comparison of T-HCTZ (10 −6 M) and NaCl with NaCl alone. www.nature.com/scientificreports/ as to the observed clinical benefit of the use of this diuretic in MD. In cardiac disease, use of diuretics has been observed to modulate an effect on pro-inflammatory cytokine release 52,53 . It is still unknown, and worthy of further exploration, as to whether during a MD attack, whether sodium dietary load alone is sufficient to trigger IL-1β processing in susceptible patients and whether these patients exhibit differential sensitivity to diuretics.  Reagents. Triamterene-hydrochlorothiazide (T-HCTZ), were dissolved in 1 ml DMSO and stock concentration of combination was 1 molar. The combination was vortexed for 3-5 min to create a homogenous solution. An equal amount of DMSO (Sigma-Aldrich) was used in the unstimulated condition, and other conditions to correct for the DMSO effect. Lipopolysaccharide (LPS) (Sigma-Aldrich) was used at 1 µg/ml as a positive control. To rule out any role of endotoxin in the expression of proinflammatory cytokines in response to NaCl, we used endotoxin-free cell-culture grade NaCl (Sigma-Aldrich, catalogue number S5886-500G) throughout our studies. Anakinra was purchased from Swedish Orphan Biovitrum AB (Sobi™).
Culture and stimulation of PBMC. Heparinized blood was obtained, and peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll/Paque gradient as describe before 37  ELISA. IL-1β and IL-6 measurements were made in the cell supernatants, by using Quantikine Human IL-1β/ IL-1F2 ELISA Kit and Human IL-6 Quantikine ELISA Kit (R&D Systems) following manufacturer's instructions. Samples were quantified using corrected values of 450 and 570 nm, reading absorbance in a microplate reader (Thermo Fisher Scientific, accuSkan). A 4-parameter logistic curve was used to describe the data. For each experiment at least 2 replicates were used for analysis.
Western blot. Cell lysates were prepared as previously described 25 . The protein content of the cell lysates was determined by bicinchoninic acid (BCA) method (Thermo Fisher Scientific). Protein (20 μg) per lane was electrophoresed through a 12% polyacrylamide gel (BIORAD) and electroblotted to a polyvinylidene difluoride (PVDF) membrane (BIORAD). Membranes were blocked with 5% non-fat dry milk (NFDM) (BIORAD) for 1 h, and then incubated with anti-IL-1β (R&D Systems) antibodies followed by horseradish peroxidase-conjugated mouse IgG (R&D Systems). Respective blots were also probed with anti-β-actin (clone AC-15, Millipore Sigma) antibody as loading controls. Enhanced chemiluminescence (Thermo Fisher Scientific) was used to visualize reactive protein bands on X-ray film. Densitometric analysis of Western blots was conducted using ImageJ software (National Institutes of Health). www.nature.com/scientificreports/